Download Confocal Laser Scanning Microscopy

University of New Hampshire
Confocal Imaging Center
Rudman Hall Room 340, 862-0632
Confocal Laser Scanning Microscopy
Exciting, State-of-the-Art Equipment Used in Many Disciplines
Biological Research:
Fixed or Living
Wholemounts, Individual Cells, Sections
A. Localization and co-localization of molecules
of interest including specific nucleotide
sequences (fluorescence in situ hybridization –
FISH) and proteins
FISH of rat hypothalamic supraoptic nucleus sections
hybridized with FITC-labeled peptidyl-glycine -amidating
monooxygenase anti-sense probe (1), TRITC-labeled
arginine vasopressin anti-sense probe (2), or both probes
(3) showing co-localization (green and red co-localization
appears yellow). From Grino & Zamora (1998) J.Histochem.
Cytochem. 46:753-759.
B. Determination of molecular proximity
otherwise well beyond the resolution of light
microscopy using FRET (fluorescence resonance
energy transfer)
Single RNA molecules labeled with a FRET donor dye and
acceptor dye in exon sequences on either side of an intron.
The splicing reaction that removes the intron sequence
requires magnesium and the protein cofactor CBP2. When
donor and acceptor dyes are relatively distant, as when the
intron is unfolded, only the donor dye emits light (green).
When donor and acceptor dyes are close (2-6 nm), as when
the intron is folded, energy transfer from donor to acceptor
results in only the acceptor dye emitting light (red). From
Zhuang Lab, Harvard University.
C. Morphogenetic and anatomical investigations
Wholemount preparations of millipede embryos at
successive stages of development, stained with actinspecific rhodamine-labeled phalloidin. Whole embryos (AE) and the first 2-3 legs (F-J) are shown. Such preparations
were used to study neurogenesis in myriapods as compared
with neurogenesis in insects and chelicerates. From Dove &
Stollewerk (2003) Development 130:2161-2171.
D. Molecular or cellular dynamics studied by
FRAP
(fluorescence
recovery
after
photobleaching) with living cells
COS-7 cells expressing a vesicular stomatitis virus G
protein-green fluorescent protein (VSVG-GFP) chimaera.
The VSVG-GFP is retained in endoplasmic reticulum (ER)
membranes. Irreversible photobleaching of the GFP within
the boxed regions (middle photos), followed by a 5 min
recovery period, demonstrates that VSVG-GFP is very
mobile within ER membranes (top panel), though not when
tunicamycin is present (bottom panel). From Nehls et al.
(2000) Nat. Cell Biol. 2:288-295.
FRAP on a biofilm of 2 strains of Pseudomonas aeruginosa,
a wild type labeled with yellow fluorescent protein and a
pilA mutant labeled with cyan fluoresecent protein (A).
After photobleaching a swath through a mixed colony (B),
only wild type bacteria had migrated into the bleached
region after 40 min (C), indicating that pilA is required for
motility. From Palmer et al. (2006) Handbook of Biological
Confocal Microscopy 3rd ed. (ed. J.B. Pawley) pp. 870-888.
Materials Research
Morphology changes during the curing of a mixture of
bisphenol-A diglycidyl ether epoxy resin and poly(3-aminopropylmethylsiloxane). All images at same magnification.
From Cabanelas et al. (2005) Polymer 46:6633-6639.
Microcircuit Research
Dried emulsion of bitumen (petroleum product component
of asphalt – dark droplets) and latex polymer (fluorescent
matrix). Taken from a study interested in producing more
resistant and environmentally-friendly asphalt (Forbes et
al. (2001) J. Microsc. 204:252-257).
Demixing of a colloid-polymer mixture (fluorescent
poly(methylmethacrylate) spheres and polystyrene,
respectively) in decalin at 6 s (a), 15 s (b), 50 s (c), and 102
s (d) after homogenization. Insets are the discrete Fourier
transforms. From Aarts & Lekkerkerker (2004) J. Phys.:
Condens. Matter 16:S4231-S4242.
Portion of an
integrated circuit showing damage (near center) resulting
from electrical overstress. The same region is shown in both
a single z-plane image and a 3-D reconstruction from a zstack of images. From Miranda & Saloma (2003) Applied
Optics
42:6520-6524.
And this is just the tip of the iceberg!